The enteric nervous system (ENS) is the largest and most complex division of the peripheral nervous system. The ENS is uniquely able to function without input from the CNS. We have demonstrated that enteric neurons are born (undergo terminal mitosis) in a reproducible order in which early-born mature neurons coexist with and innervate still-dividing precursors. Serotonergic and cholinergic neurons are born first, while neurons that contain tyrosine hydroxylase (TH), g-aminobutyric acid (GABA), or calcitonin gene related peptide (CGRP) are born later. This observation led us to frame the hypothesis that the activity of early-born neurons can, through their neurotransmitters, 5-HT and/or acetylcholine (ACh), affect the development of later-born neurons. Supporting of this idea, we showed that 5-HT, through 5-HT4 receptors, promotes development of TH-, GABA-, and CGRP-containing neurons, that these phenotypes are deficient, and the ENS is hypoplastic when tryptophan hydroxylase 2 (TPH2) is deleted and mice thus lack neuronal 5-HT. The late-born neurons are also deficient and the ENS is hypoplastic in animals that carry an autism-associated human variant of the serotonin transporter (SERT; SERT Ala56 or G56A), which is hyperfunctional and clears 5-HT from its receptors too rapidly. In contrast, mice that lack SERT (SERTKO) or which are exposed during development to a SERT inhibitor have a hyperplastic ENS and excessive numbers of late-born neurons. Recent preliminary data, obtained with mice that under- or overexpress the presynaptic choline transporter, suggest that ACh functions like 5-HT. Because serotonergic and cholinergic neurons are thus essential for ENS development, defects in their signaling during ontogeny lead, not only to ENS hypo-or hyperplasia, but to dysmotilities and abnormally regulated mucosal growth that are readily demonstrated in adult animals. We thus postulate that the defects that arise due to errant serotonergic or cholinergic signaling in ontogeny, possibly due to environmental perturbations, contribute to dysmotility disorders in adults. Although TPH2-derived 5-HT is more important than that from TPH1 in ENS formation under basal conditions, TPH1-derived 5-HT from ?pre-enteric? sources, (placenta, yolk sac, and maternal circulation) may be essential to support ENS neurogenesis prior to development of serotonergic neurons. TPH1-derived 5-HT from mucosal enterochromaffin (EC) cells may also disturb ENS neurogenesis and/or function if it reaches the neuronal compartment. We now plan to test 3 overarching hypotheses: (i) ?Pre-enteric? TPH1-derived 5-HT is essential to support enteric neurogenesis before serotonergic neurons develop. (ii) Mucosal SERT activity is essential to prevent 5-HT from overflowing from the mucosa to disturb neurogenesis and/or neuronal function; insults that up- or downregulate SERT thus cause abnormal ENS formation and adult function. (iii) Early-born enteric cholinergic neurons act on muscarinic receptor(s) to stimulate the generation of late-born enteric neurons and also are essential for normal ENS development.